The completion of the Human Genome Project has cast a spotlight on gene therapy to potentially treat numerous diseases. Viral-based gene delivery approaches, while powerful, have generated many concerns regarding adverse effects [1, 2]. By contrast, non-viral gene delivery is regarded as safer, although due to its broad definition it encompasses many different strategies and materials [3,4]. This non-viral approach generally relies on a polycation to (partially) mimic the role of a viral capsid. Unfortunately, the gains from various non-viral formulations are not yet sufficient to satisfy both efficiency and toxicity requirements. Based on our knowledge of self-assembly, and the ability to "program" DNA into nanostructures, it is natural to ask if these nanostructures can be alternatives to carrier-based non-viral gene delivery. Our hypothesis is that DNA nanostructures can demonstrate intracellular antisense function and thus serve as a new modality in non-viral gene delivery. This proposal is divided into two aims. Aim 1 will entail design of DNA nanostructures with antisense capabilities for intra-cellular activity. We will also tune the bioactivity of the antisense features by changing the structural features of the DNA objects. Aim 2 will entail the creation of DNA nanostructures, and various formulations, to optimize their ability for cellular internalization using several cell types. The long-term goal of the proposed research is to explore alternatives to traditional non-viral gene delivery through the use of DNA nanostructures that are resistant to enzymatic degradation, have antisense properties, and can be internalized by cells. The proposed approach is likely to contribute to our fundamental understanding of the barriers in non-viral gene delivery, in addition to promising a potentially new modality for therapy. PUBLIC HEALTH RELEVANCE: The treatment of diseases through gene therapy continues to be a long-sought goal. The development of new approaches to non-viral gene delivery is therefore of great relevance to human health. By using DNA nanostructures as the main component of a gene delivery vehicle, we are proposing a fundamentally different approach that may ultimately prove more efficient and safe.